Work continued to understand the genetic regulation of expression of proteins secreted by Bacillus anthracis and Bacillus cereus that are potential virulence factors. Proteins of particular interest are hemolysins, toxins, and proteases. In B. cereus, expression and secretion of these is controlled by a global regulator, PlcR, which is part of a quorum sensing system that responds to peptides generated from a small protein, PapR. During this period, work focused on identifying the secreted protease that is responsible for generation of the peptides from PapR. A candidate protease designated NprB was identified by genomic comparisons and it was inactivated using the Cre-Lox system developed previously for gene knockouts. The NprB gene KO eliminated the quorum sensing system, as reflected in the loss of expression of several secreted factors. In other work directed toward resolving a perennial question in anthrax genomics, we systematically deleted portions of the large virulence plasmid pXO1 to identify a minimal replicon, the smallest circular DNA sequence region able to replicate independently within B. anthracis. Large deletions extending over 10-20 genes within pXO1 were generated using an improved and simplified version of the Cre recombinase-based system. This work succeeded in identifying a small region including just two genes, ORFs 14 and 16, as being essential for replication. The new Cre-loxP system was also used to delete additional secreted proteases. One of these proteases, the secreted zinc metalloprotease InhA1, was shown in a collaborative effort to play an essential role in activation of prothrombin and factor X in a unique microfluidics-based analytic system. In a separate effort, work is progressing to improved tools for controlling and detecting heterologous gene expression in B. anthracis. A series of codon-optimized fluorescent protein genes were developed for use in Gram-positive bacteria with low GC content, such as B. anthracis. These proved to be very effective for monitoring promoter activity in this pathogen. Following publication of this result, more than twenty laboratories have requested these fluorescent protein plasmids for use in a variety of Gram-positive pathogens. During this period we also continued to promote the use of a unique monoclonal antibody, S9.6, that reacts in a sequence-independent manner with DNA/RNA hybrids. Several labs to which we have supplied this antibody have confirmed that it has great value in development of microarrays, in part because it eliminates the need to label or amplify cellular RNAs, procedures that can introduce bias or variation. Work is now underway to engineer this antibody into other protein constructs so as to extend its utility in several research and diagnostic applications.